Lateral flow blotting assay

ABSTRACT

Methods, compositions, and kits for performing analyte detection in a lateral flow assay.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.14/634,209, filed Feb. 27, 2015, which claims priority to U.S.Provisional Application No. 61/945,376, filed Feb. 27, 2014, thecontents of both which are hereby incorporated by reference in theirentireties for all purposes.

BACKGROUND OF THE INVENTION

Methods for detection of immobilized analytes are commonly employed inthe biological sciences. For example, traditional blotting (e.g.,Southern, northern, western, far western, eastern, vacuum, middleeastern, eastern-western, and far-eastern blotting, etc.) can be used todetect analytes immobilized on a substrate or membrane or in a matrix(e.g., in agarose or acrylamide). In general, such blotting techniquesinvolve immobilization of the analyte(s) to be detected and contactingthe analyte(s) with a binding reagent (e.g., an antibody). Blotting alsousually involves multiple washing steps and/or blocking steps betweenimmobilization and final detection. Such washing and blocking stepsconsume a practitioner's limited time and/or reagents and are a frequentsource of error and irreproducibility.

BRIEF SUMMARY OF THE INVENTION

The present invention provides improved methods, compositions, and kitsfor detection of immobilized analytes.

In some embodiments, the present invention provides a porous substratehaving a length and a width and comprising a reagent reservoir regionand a lateral flow region, said reagent reservoir region comprising orbordered by an impermeable or hydrophobic barrier, said impermeable orhydrophobic barrier substantially blocking flow of a liquid from thereagent reservoir region into the lateral flow region until lateral flowis initiated. In some cases, the lateral flow region is configured towick one or more binding reagents from the reagent reservoir and alongthe length of the lateral flow region after contacting the lateral flowregion with the reagent reservoir region.

In some embodiments, the porous substrate further comprises a foldingregion, wherein the folding region is positioned to allow initiating oflateral flow by folding the porous substrate at the folding region,wherein folding the porous substrate at the folding region contacts atleast a portion of the reagent reservoir region to at least a portion ofthe lateral flow region.

In some aspects, said reagent reservoir region comprises at least afirst reagent reservoir sub-region and a second reagent reservoirsub-region, wherein the first and second reagent reservoir sub-regionsare not in fluid communication; and said lateral flow region comprisesat least a first lateral flow sub-region and a second lateral flowsub-region, wherein said first and second lateral flow sub-regions arenot in fluid communication.

In some cases, the first reagent reservoir sub-region is separated fromthe second reagent reservoir sub-region by a hydrophobic or impermeablebarrier. In some cases, the first lateral flow sub-region is separatedfrom the second lateral flow sub-region by a hydrophobic or impermeablebarrier. In some cases, each reagent reservoir sub-region is configuredsuch that each sub-region can be separately contacted to a correspondinglateral flow sub-region. For example, one or more reagent reservoirsub-regions can be individually and independently folded at the foldingreagent to contact a corresponding lateral flow sub-region.

In some embodiments, the lateral flow region and/or the reagentreservoir region comprises a capillary flow matrix. In some aspects, atleast a portion of the porous substrate, e.g., at least a portion of thecapillary flow matrix, is coupled to an impermeable or hydrophobicbacking. The capillary flow matrix can comprise cellulose or glassfibers.

In some embodiments, the folding region comprises a visual marker, apleat, or a crease. In some cases, the folding region extends across thewidth of the porous substrate. In some cases, the lateral flow region isconfigured to wick one or more binding reagents from the reagentreservoir and along the length of the lateral flow region after foldingthe folding region.

In some embodiments, at least a portion of the porous substrate issubstantially compressible. For example, in some cases, at least aportion of the lateral flow region is substantially compressible. Asanother example, at least a portion of the reagent reservoir region issubstantially compressible.

In some embodiments, the reagent reservoir region comprises a firstprimary binding reagent. The reagent reservoir region can also comprisea second primary binding reagent. In some aspects, the first and secondprimary binding reagents of the reagent reservoir region are separatedby an impermeable or hydrophobic barrier.

In any of the preceding aspects, embodiments, or cases, the reagentreservoir region and/or the lateral flow region of the porous substratecan contain a protein aggregation modifying agent.

In some embodiments, the present invention provides a method forperforming a lateral flow assay comprising: —providing a poroussubstrate comprising a lateral flow region; —placing the poroussubstrate in intimate contact with a membrane comprising a plurality ofimmobilized analytes; and —contacting the lateral flow region with areagent reservoir region comprising a primary binding reagent, therebycausing at least a portion of the primary binding reagent to wick intothe lateral flow region, wherein causing at least a portion of theprimary binding reagent to wick into the lateral flow region initiateslateral flow of the primary binding reagent through at least a portionof the lateral flow region, thereby contacting at least a portion of theprimary binding reagent to the membrane comprising a plurality ofimmobilized analytes.

In some aspects, the method further comprises initiating lateral flow ofa wash solution through at least a portion of the lateral flow region.In some aspects, the method further comprises initiating lateral flow ofa solution containing a secondary binding reagent through at least aportion of the lateral flow region. In some cases, initiating lateralflow of a wash solution can be performed after initiating lateral flowof the solution containing a secondary binding reagent through at leasta portion of the lateral flow region. In some cases, initiating lateralflow of a wash solution can be performed after lateral flow of theprimary binding reagent.

In some aspects, the porous substrate comprising a lateral flow regionfurther comprises the reagent reservoir region, and the reagentreservoir region is separated from the lateral flow region by (i) animpermeable or hydrophobic barrier and (ii) a folding region. In somecases, wherein the folding region extends across the width of the poroussubstrate. In some cases, contacting the porous substrate with thereagent reservoir region comprising the primary binding reagentcomprises folding the porous substrate at the folding region. In somecases, the lateral flow region is configured to wick one or more bindingreagents from the reagent reservoir and along the length of the lateralflow region after folding the folding region. The folding region cancomprise a visual marker, a pleat, or a crease. In some cases, at leasta portion of the lateral flow region, at least a portion of the reagentreservoir region, or at least a portion of the porous substrate issubstantially compressible.

In some embodiments, the reagent reservoir region comprises at least afirst reagent reservoir sub-region and a second reagent reservoirsub-region, wherein the first and second reagent reservoir sub-regionsare not in fluid communication; and said lateral flow region comprisesat least a first lateral flow sub-region and a second lateral flowsub-region, wherein said first and second lateral flow sub-regions arenot in fluid communication. In some aspects, the first and secondreagent reservoir sub-regions can be separately contacted with the firstand second lateral flow sub-regions respectively. For example, one ormore reagent reservoir sub-regions can each be independently folded atthe folding reagent to each contact a corresponding lateral flowsub-region.

In some cases, the first reagent reservoir sub-region is separated fromthe second reagent reservoir sub-region by a hydrophobic or impermeablebarrier, and the first lateral flow sub-region is separated from thesecond lateral flow sub-region by a hydrophobic or impermeable barrier.

In some embodiments, the first reagent reservoir sub-region comprises afirst primary binding reagent and the second reagent reservoirsub-region comprises a second primary binding reagent. In someembodiments, the reagent reservoir region comprises a proteinaggregation modifying agent.

In some embodiments of the method, the porous substrate, at least aportion of the porous substrate, the reagent reservoir region, at leasta portion of the reagent reservoir region, the lateral flow region,and/or at least a portion of the lateral flow region comprises acapillary flow matrix. For example, the capillary flow matrix cancomprise cellulose or glass fibers. In some embodiments of the method,the porous substrate, at least a portion of the porous substrate, thereagent reservoir region, at least a portion of the reagent reservoirregion, the lateral flow region, and/or at least a portion of thelateral flow region is coupled to an impermeable or hydrophobic backing.

In some embodiments, the present invention provides a kit comprising atleast one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of any of theforegoing porous substrates. In some cases, the kit further comprises afirst and a second primary binding reagent. In some cases, the kitfurther comprises a container having a protein aggregation modifyingagent. In some cases, the protein aggregation modifying agent is acyclodextrin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a porous substrate with three separate reagent reservoirsub-regions and three separate corresponding lateral flow sub-regions.The sub-regions are all separated by hydrophobic barriers so that thereis no flow of binding reagents between sub-regions. The reagentreservoir sub-regions are loaded with different binding reagents toenable detection of multiple analytes.

FIG. 2 depicts folding of the porous substrate at the folding region sothat the reagent reservoir region contacts the lateral flow sub-region,thereby initiating lateral flow of reagents in the direction indicatedby the arrows. The hydrophobic barriers block flow of reagents frombetween lateral flow sub-regions during lateral flow.

FIG. 3 depicts a method of performing multiplex detection of analytes ona western blot membrane using a porous substrate described herein. Aporous substrate is provided and placed in intimate contact with awestern blot membrane containing bound proteins (analytes).

In step 1, binding reagents (e.g., antibodies) are loaded intoself-contained reagent reservoir sub-regions demarcated by hydrophobicbarriers. In step 2, the porous substrate is folded to cause contactbetween the reagent reservoir and lateral flow region. Wicking causeslateral flow of the antibodies through the porous substrate, therebycontacting the antibodies to the proteins bound to the western blotmembrane. The immobilized proteins indicated by the circles are detectedand identified by their binding to a binding reagent. The location ofthe detected analyte on the membrane can provide further informationregarding the identity of the analyte.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “analyte” refers to a biological molecule, e.g., a protein,nucleic acid, polysaccharide, lipid, antigen, growth factor, hapten,etc., or a portion thereof. Analytes can be irreversibly immobilized ona surface, such as a membrane and detected as described herein.

The term “immobilized” as used herein refers to reversibly andirreversibly immobilized molecules (e.g., binding reagents or analytes).Reversibly immobilized molecules are immobilized in a manner that allowsthe molecules, or a portion thereof (e.g., at least 25%, 50%, 60%, 75%,80% or more of the molecules), to be removed from their immobilizedlocation without substantial denaturation or aggregation. For example, amolecule can be reversibly immobilized in or on a porous substrate bycontacting a solution containing the molecule with the porous substrate,thereby soaking up the solution and reversibly immobilizing themolecule. The reversibly immobilized molecule can then be removed bywicking the solution from the porous substrate, or from one region ofthe porous substrate to another. In some cases, a molecule can bereversibly immobilized on a porous substrate by contacting a solutioncontaining the molecule with the porous substrate, thereby soaking upthe solution, and then drying the solution containing porous substrate.The reversibly immobilized molecule can then be removed by contactingthe porous substrate with another solution of the same or a differentcomposition, thereby solubilizing the reversibly immobilized molecule,and then wicking the solution from the porous substrate, or from oneregion of the porous substrate to another.

Irreversibly immobilized molecules (e.g., binding reagents or analytes)are immobilized such that they are not removed, or not substantiallyremoved, from their location under mild conditions (e.g., pH betweenabout 4-9, temperature of between about 4-65° C.). Exemplaryirreversibly immobilized molecules include protein analytes bound to anitrocellulose or polyvinylidene fluoride membrane by standard blottingtechniques (e.g., electroblotting).

The term “binding reagent” refers to a reagent that specifically bindsto a molecule such as an analyte. A wide variety of binding reagents areknown in the art, including antibodies, aptamers, affimers, lipocalins(e.g., anticalins), thioredoxin A, bilin binding protein, or proteinscontaining an ankyrin repeat, the Z domain of staphylococcal protein A,or a fibronectin type III domain.

The term “specifically bind” refers to a molecule (e.g., binding reagentsuch as an antibody or antibody fragment) that binds to a target with atleast 2-fold greater affinity than non-target compounds, e.g., at least4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,25-fold, 50-fold, 100-fold, or 1000-fold or more greater affinity.

The term “antibody” refers to a polypeptide comprising a frameworkregion from an immunoglobulin gene, or fragments thereof, thatspecifically bind and recognize an antigen, e.g., a particular analyte.Typically, the “variable region” contains the antigen-binding region ofthe antibody (or its functional equivalent) and is most critical inspecificity and affinity of binding. See Paul, Fundamental Immunology(2003).

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

An “isotype” is a class of antibodies defined by the heavy chainconstant region. Immunoglobulin genes include the kappa, lambda, alpha,gamma, delta, epsilon, and mu constant region genes. Light chains areclassified as either kappa or lambda. Heavy chains are classified asgamma, mu, alpha, delta, or epsilon, which in turn define the isotypeclasses, IgG, IgM, IgA, IgD and IgE, respectively.

Antibodies can exist as intact immunoglobulins or as any of a number ofwell-characterized fragments that include specific antigen-bindingactivity. Such fragments can be produced by digestion with variouspeptidases. Pepsin digests an antibody below the disulfide linkages inthe hinge region to produce F(ab)′₂, a dimer of Fab which itself is alight chain joined to V_(H)—C_(H)1 by a disulfide bond. The F(ab)′₂ maybe reduced under mild conditions to break the disulfide linkage in thehinge region, thereby converting the F(ab)′₂ dimer into an Fab′ monomer.The Fab′ monomer is essentially Fab with part of the hinge region (seeFundamental Immunology (Paul ed., 3d ed. 1993). While various antibodyfragments are defined in terms of the digestion of an intact antibody,one of skill will appreciate that such fragments may be synthesized denovo either chemically or by using recombinant DNA methodology. Thus,the term antibody, as used herein, also includes antibody fragmentseither produced by the modification of whole antibodies, or thosesynthesized de novo using recombinant DNA methodologies (e.g., singlechain Fv) or those identified using phage display libraries (see, e.g.,McCafferty et al., Nature 348:552-554 (1990)).

A “monoclonal antibody” refers to a clonal preparation of antibodieswith a single binding specificity and affinity for a given epitope on anantigen. A “polyclonal antibody” refers to a preparation of antibodiesraised against a single antigen, but with different bindingspecificities and affinities.

As used herein, “V-region” refers to an antibody variable region domaincomprising the segments of Framework 1, CDR1, Framework 2, CDR2, andFramework 3, including CDR3 and Framework 4, which segments are added tothe V-segment as a consequence of rearrangement of the heavy chain andlight chain V-region genes during B-cell differentiation.

As used herein, “complementarity-determining region (CDR)” refers to thethree hypervariable regions in each chain that interrupt the four“framework” regions established by the light and heavy chain variableregions. The CDRs are primarily responsible for binding to an epitope ofan antigen. The CDRs of each chain are typically referred to as CDR1,CDR2, and CDR3, numbered sequentially starting from the N-terminus, andare also typically identified by the chain in which the particular CDRis located. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found.

The sequences of the framework regions of different light or heavychains are relatively conserved within a species. The framework regionof an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three dimensional space.

The amino acid sequences of the CDRs and framework regions can bedetermined using various well known definitions in the art, e.g., Kabat,Chothia, international ImMunoGeneTics database (IMGT), and AbM (see,e.g., Johnson et al., supra; Chothia & Lesk, (1987) J Mol. Biol. 196,901-917; Chothia et al. (1989) Nature 342, 877-883; Chothia et al.(1992) J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J.Mol.Biol 1997,273(4)). Definitions of antigen combining sites are also described inthe following: Ruiz et al. Nucleic Acids Res., 28, 219-221 (2000); andLefranc Nucleic Acids Res. January 1; 29(1):207-9 (2001); MacCallum etal., J. Mol. Biol., 262: 732-745 (1996); and Martin et al, Proc. NatlAcad. Sci. USA, 86, 9268-9272 (1989); Martin, et al, Methods Enzymol.,203: 121-153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992); andRees et al, In Sternberg M. J. E. (ed.), Protein Structure Prediction.Oxford University Press, Oxford, 141-172 1996).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region, CDR, or portion thereof) islinked to a constant region of a different or altered class, effectorfunction and/or species, or an entirely different molecule which confersnew properties to the chimeric antibody (e.g., an enzyme, toxin,hormone, growth factor, drug, etc.); or (b) the variable region, or aportion thereof, is altered, replaced or exchanged with a variableregion having a different or altered antigen specificity (e.g., CDR andframework regions from different species).

The antibody binds to an “epitope” on the antigen. The epitope is thespecific antibody binding interaction site on the antigen, and caninclude a few amino acids or portions of a few amino acids, e.g., 5 or6, or more, e.g., 20 or more amino acids, or portions of those aminoacids. In some cases, the epitope includes non-protein components, e.g.,from a carbohydrate, nucleic acid, or lipid. In some cases, the epitopeis a three-dimensional moiety. Thus, for example, where the target is aprotein, the epitope can be comprised of consecutive amino acids, oramino acids from different parts of the protein that are brought intoproximity by protein folding (e.g., a discontinuous or conformationalepitope). The same is true for other types of target molecules that formthree-dimensional structures.

II. Introduction

Compositions, methods, and kits described herein can be useful fordetecting analytes bound to a membrane. For example, described herein isa porous substrate. The porous substrate can be placed in intimatecontact with a membrane containing bound analytes. Lateral flow of oneor more binding reagents through the region of the porous substrate inintimate contact with the membrane can allow the one or more bindingreagents to bind, and thereby detect, one or more membrane-boundanalytes. The porous substrate can be divided into two or moresub-regions by impermeable or hydrophobic barriers such that detectionof one or more analytes in one sub-region does not confound detection inanother sub-region. Thus, multiplex detection can be performed using thecompositions, methods, and kits described herein. In some cases, theporous substrate is configured for use in a sequential lateral flowdevice.

III. Compositions

A. Porous Substrate

A porous substrate can useful for storage of one or more bindingreagents. A porous substrate can also be useful for detection of one ormore immobilized analytes. The porous substrate has a width, a length,and a height (e.g., a thickness). The substrate can be any size andshape. In certain embodiments, the porous substrate is planar, e.g., theporous substrate can approximate or be a rectangular plane. In somecases, the length and the width of the porous substrate are at leastabout 2-fold, 5-fold, 10-fold, 100-fold or more larger than the height(i.e., thickness). In some embodiments, the porous substrate is sizedfor use in a blotting apparatus.

For example, the porous substrate can be sized to transfer reagentsthrough at least a portion of the porous substrate, and thereby contactthe reagents to a blotting membrane having one or more analytesirreversibly immobilized thereon. In some embodiments, the poroussubstrate has an impermeable, or substantially impermeable backing.

Exemplary sizes for porous substrates include, without limitation,porous substrates that are at least about 0.25 cm, 0.5 cm, 1 cm, 2 cm, 3cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 10 cm, 12 cm, 15 cm, 20 cm, 30 cm ormore in at least one dimension. Exemplary sizes of rectangular planarporous substrates include porous substrates that are about 1 cm×1 cm,7×8.4 cm, 8.5×13.5 cm, 10 cm×15 cm, or 25×28 cm in length and widthrespectively. Exemplary sizes further include 8.5 cm×9 cm, 7 cm×9 cm, 8cm×10.7 cm, 10 cm×10 cm, 7 cm×8.5 cm, 8.3 cm×7.3 cm, 8 cm×8 cm, 8.3cm×13 cm, 10.8 cm×13.5 cm. In some embodiments, the porous substrate is18 cm in length by 10 cm in width. In some cases, the porous substrateis 18±0.5, 1, 2, or 3 cm in length by 10±0.5, 1, 2, or 3 cm in width.

In some embodiments, the porous substrate is configured to have a highsolution capacity and lateral flow rate. In some cases, the highsolution capacity and lateral flow rate are provided by having a poroussubstrate with substantial height (e.g., thickness). In some cases, theporous substrate is about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, orabout 0.2 mm thick. In some cases, the porous substrate is between about0.05 mm and about 0.5 mm thick.

Porous substrates generally have a large surface area due to thepresence of a plurality of pores. The large surface area can increasethe loading capacity of the porous substrate for one or more reagents orone or more solutions containing a reagent. In some embodiments, theporous substrate, or a portion thereof, such as a lateral flow region ora reagent reservoir region, has a large surface area as compared to anonporous substrate of the same material and size. For example, theporous substrate can have at least about a 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 40, 50, 100, 200, 300, 500, 1000-fold or more increasedsurface area as compared to a nonporous substrate of the same materialand size. In some embodiments, the porous substrate, or a portionthereof, such as a lateral flow region or a reagent reservoir region,has a large specific surface area. For example, the porous substrate canhave a specific surface area of at least about 0.1 m²/g, 0.5 m²/g, 1m²/g 10 m²/g, or more as measured by standard techniques.

In some embodiments, the porous substrate, or a portion thereof, such asa lateral flow region or a reagent reservoir region, possesses a highspecific binding capacity for a binding reagent (e.g., antibody). Forexample, in some cases, the porous substrate can reversibly immobilizeat least about 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 40 mg,60 mg, 100 mg, or more binding reagent for every mg of substratematerial.

In some embodiments, the porous substrate can have a particular poresize, a particular average pore size, or a particular pore size range.For example, the porous substrate can contain 0.1 μm pores, 0.2 μmpores, 0.45 μm pores, or 1, 2, 4, 5, 6, 7, 8, 10, 15, 20 μm pores, orpores larger than about 20 μm. As another example, the porous substratecan contain pores that average 0.1, 0.2, 0.45, 1, 2, 4, 5, 6, 7, 8, 10,15, or 20 μm, or more in size. As another example, the porous substratecan contain pores that range about 0.1-8 μm, 0.2-8 μm, 0.45-8 μm, 1-8μm, 0.1-4 μm, 0.1-2 μm, 0.1-1 μm, 0.1-0.45 μm, 0.2-8 μm, 0.2-4 μm, 0.2-2μm, 0.2-1 μm, 0.2-0.45 μm, 0.45-8 μm, 0.45-4 μm, 0.45-2 μm, 0.45-1 μm insize. In some cases, the porous substrate can contain pores that areless than about 20 μm in size. For example, the porous substrate can becomposed of a material in which at least about 50%, 60%, 70%, 80%, 90%or more of the pores are less than about 20, 15, 10, or 5 μm in size. Insome cases, the pores are large enough to contain one or more proteinsof average size (e.g., about 1 nm). For example, the pores can be atleast 1 nm in size, at least 5 nm in size, at least 10, 100, or 500 nmin size. Alternatively, at least 50%, 60%, 70%, 80%, 90% or more of thepores can be more than 1, 5, 10, 50, 100, or 500 nm in size. As usedherein, pore size can be measured as a radius or a diameter. In somecases, the porous substrate contains porous polyethylene, such as porouspolyethylene having a pore size between 0.2 and 20 microns, or between 1and 12 microns. The porous substrate can have a different pore size indifferent regions of the substrate. For example, a reagent reservoirregion can have a pore size or pore size range, and a lateral flowregion can have a different pore size or pore size range.

The substrate can be treated or functionalized to minimize non-specificreagent binding, increase lateral flow, increase wicking, or to reduceprotein aggregation. For example, the substrate, or a portion thereof,can be treated to alter the hydrophilicity or alter the hydrophobicityof the treated area. In some cases, altering the hydrophilicity orhydrophobicity of a porous substrate can increase binding reagentloading, decrease binding reagent aggregation or denaturation, createmask regions in which binding reagent is excluded from or not loaded, ordirect flow of binding reagents when the substrate is wet. In somecases, the porous substrate contains a protein aggregation modifyingagent as described herein.

The porous substrate can be marked or annotated such that the origin,composition, or location of a reversibly immobilized binding reagent(e.g., a primary antibody) is recorded. For example, one or more regionscontaining reversibly immobilized binding reagent(s) can be visuallydiscernible, such that one of skill in the art can determine thelocation of the reversibly immobilized binding reagent. In some cases,the name of the binding reagent (e.g., anti-phospho PIK3), identity(e.g., catalog number), amount, lot number, etc. can be printed,stamped, or otherwise indicated on a portion of the porous substrate. Insome cases, the substrate is marked or annotated such that the properorientation for use in a blotting device, e.g., in a sequential lateralflow capillary device, is discernible.

Porous substrates can include, but are not limited to, polymercontaining substrates. The polymer can be in the form of polymer beads,a polymer membrane, or a polymer monolith. In some cases, the polymer iscellulose. Cellulose containing substrates include paper, cloth, woven,or non-woven cellulose substrates. Cloth substrates include thosecontaining a natural cellulose fiber such as cotton or wool. Papersubstrates include those containing natural cellulose fiber (e.g.,cellulose or regenerated cellulose) and those containing cellulose fiberderivatives including, but not limited to cellulose esters (e.g.,nitrocellulose, cellulose acetate, cellulose triacetate, celluloseproprionate, cellulose acetate propionate, cellulose acetate butyrate,and cellulose sulfate) and cellulose ethers (e.g., methylcellulose,ethylcellulose, ethyl methyl cellulose, hydroxyethyl cellulose,hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, ethylhydroxyethyl cellulose, and carboxymethyl cellulose). In some cases, thecellulose substrate contains rayon. In some cases, the substrate ispaper, such as a variety of WHATMAN® paper.

Porous substrates can also include, but are not limited to, substratesthat contain a sintered material. For example, the substrate can containa sintered glass, a sintered polymer, or sintered metal, or acombination thereof. In some cases, the sintered material is formed bysintering one or more of powdered glass, powdered polymer, or powderedmetal. In other cases, the sintered material is formed by sintering oneor more of glass, metal, or polymer fibers. In still other cases, thesintered material is formed from the sintering of one or more of glass,polymer, or metal beads.

Porous substrates can also contain, but are not limited to, one or morenon-cellulosic polymers, e.g. a synthetic polymer, a natural polymer, ora semisynthetic polymer. For example, the substrate can contain apolyester, such as polyglycolide, polylactic acid, polycaprolactone,polyethylene adipate, polyhydroxylalkanoate, polyhydroxybutyrate,poly(3-hydroxybutyrate-co-3-hydroxyvalerate, polyethylene terephthalate,polybutylene terephthalate, polytrimethylene terephthalate, polyethylenenaphthalate, Vectran®. In some cases, the polymer is spunbound, such asa spunbound polyester.

Additional synthetic polymers include, but are not limited to nylon,polypropylene, polyethylene, polystyrene, divinylbenzene, polyvinyl,polyvinyl difluoride, high density polyvinyl difluoride, polyacrylamide,a (C₂-C₆) monoolefin polymer, a vinylaromatic polymer, avinylaminoaromatic polymer, a vinylhalide polymer, a (C₁-C₆) alkyl(meth)acrylate polymer, a(meth)acrylamide polymer, a vinyl pyrrolidonepolymer, a vinyl pyridine polymer, a (C₁-C₆) hydroxyalkyl (meth)acrylatepolymer, a (meth)acrylic acid polymer, an acrylamidomethylpropylsulfonicacid polymer, an N-hydroxy-containing (C₁-C₆) alkyl(meth)acrylamidepolymer, acrylonitrile or a mixture of any of the foregoing.

Porous substrates can also contain, but are not limited to, one or morepolysaccharides. Exemplary polysaccharides include those containingcellulose, agarose, amylose, chitin, chitosan, galactosamine, curdlan,dextran, xylan, inulin, and derivatives thereof, e.g., esters, phenylcarbamates, alkyl carabmates, and benzyl carbamates. In some cases, thepolysaccharides are cross-linked. For example, the porous substrate caninclude agarose, or a cross linked agarose. In some cases, the substratecan include cross links between the polysaccharide and otherconstituents of the substrate.

Porous substrates also include, but are not limited to, capillarywicking beds and materials used therein. For example, the substrate caninclude a thin layer chromatography plate, or be formed of any of thethin layer chromatography substrates known in the art. Thin layerchromatography substrates known in the art include, but are not limitedto, silica, silica derivatized with C₄, C_(g), or C₁₈ alkyl groups, andalumina. Porous substrates can also contain, but are not limited to,glass, glass fibers, fiberglass, natural or synthetic sponge, silica,alumina, or a derivative thereof. In some cases, the glass fiber is aglass fiber derivative. In some cases, the porous substrate containsglass fiber and another porous material such as cellulose, a cellulosederivative, and/or polyester.

In addition to the foregoing substrate materials, the substrate can alsocontain any combination of the foregoing. In some cases, the substratecan contain composite materials that include a combination of materialsdescribed above. For example, the substrate can contain glass or silicafibers in a synthetic polymer matrix. As another example, the poroussubstrate can contain plastic backed glass fiber, or plastic backednitrocellulose. Additional suitable materials that may comprise theporous substrate include any of the materials, or a combination thereof,described in Jallerat & Thom, Filter Membranes and BioseparationEquipment and Supplies, IVD Technology (October, 2004), or any of thematerials described in U.S. Pat. No. 4,632,901; or U.S. PatentApplication Nos. 2010/0239459, and 2013/0164193. In some embodiments,one or more regions of the porous substrate, or a portion thereof, issubstantially compressible. As used herein, substantially compressiblerefers to a material that retains structural integrity under an appliedpressure that compresses the material. For example, a porous substratethat is substantially compressible can be compressed along at least oneaxis such that the length of the compressed axis is reduced by at leastabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 33%,40%, 50%, 60%, 66%, 75%, 80%, 90%, or more, without loss of structuralintegrity.

In some embodiments, the porous substrate is configured to enabledetection of one or more analyte(s) by lateral flow in a lateral flowdevice (e.g., a sequential lateral flow device). For example, the poroussubstrate can be useful for contacting one or more binding reagents toone or more membrane-bound analytes by lateral flow in a sequentiallateral flow device. In some cases, the sequential lateral flow deviceis a passive sequential lateral flow device. Exemplary passivesequential lateral flow devices are described in U.S. Patent ApplicationNos. 2010/0239459, and 2013/0164193.

The porous substrate can be suitable for storage. For example, theporous substrate can be stored for at least about a day, three days,7-10 days, at least about a month, two months, 3 months, six months, ayear or longer. In some cases, the porous substrate can be stored (e.g.,at about 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 25, 30, 35, or 37°C. or more) for at least about a day, three days, 7-10 days, at leastabout a month, two months, 3 months, six months, a year or longer. Theporous substrate can be stored dry, substantially dry, or wet. In somecases, a portion of the porous substrate is stored dry or substantiallydry and a portion is stored wet. In some cases, a portion of the poroussubstrate is stored dry and a portion is stored substantially dry. Theporous substrate can be stored and then removed from storage, optionallyreconstituted, and then provided as described herein to detect one ormore membrane-bound analytes.

The porous substrates can be suitable for storage of binding reagents.For example, one or more binding reagents can be contacted with a poroussubstrate (e.g., contacted with a reagent reservoir region or subregionof a porous substrate) and the porous substrate then stored (e.g.,stored for at least about a day, three days, 7-10 days, at least about amonth, two months, 3 months, six months, a year or longer). The storedporous substrate and one or more binding reagents can then be used todetect one or more analytes bound to a membrane. In some cases, thebinding reagent is dried, or substantially dried, onto the poroussubstrate prior to storage. Dry or substantially dry binding reagentscan be reconstituted during or prior to use in detecting membrane boundanalytes by contact with an aqueous solution.

i. Reagent Reservoir Region and Lateral Flow Region

The porous substrate can have a reagent reservoir region and a lateralflow region. In some embodiments, the reagent reservoir region is areservoir for one or more binding reagents (e.g., one or more primaryand/or one or more secondary antibodies). In some embodiments, thelateral flow region is a region of the porous substrate configured towick one or more binding reagents, or a portion thereof, from thereagent reservoir region and through the lateral flow region, or aportion thereof. The reagent reservoir region of the porous substratecan be separated from the lateral flow region of the porous substrate bya barrier such that the lateral flow region does not wick solution orreagents from the reagent reservoir region until lateral flow isinitiated. Such barriers include hydrophobic or impermeable barriersthat are further described herein.

In some embodiments, the reagent reservoir region contains a reversiblyimmobilized binding reagent, such as an antibody. In some cases, thereversibly immobilized binding reagent is present on or in the reagentreservoir region in a dry or substantially dry state. In otherembodiments, the reversibly immobilized binding reagent is present on orin the reagent reservoir region in solution. In some cases, the reagentreservoir region is a sponge, adsorbant, or absorbant that reversiblycontains, or can reversibly contain, a binding reagent (e.g., insolution, dry, or substantially dry). In some embodiments, the reagentreservoir region is configured to be contacted with a solutioncontaining a binding reagent and thereby reversibly immobilize thebinding reagent. In some embodiments, the reagent reservoir region isconfigured to be contacted with a solution containing a binding reagentand then dried or substantially dried, thereby reversibly immobilizingthe binding reagent.

In some embodiments, the reagent reservoir region is composed of thesame material as the lateral flow region. In other embodiments, thereagent reservoir region is composed of a different material as comparedto the lateral flow region. For example, the reagent reservoir regionand lateral flow region can have a different pore size, thickness,composition, etc. In some cases, the reagent reservoir region is thesame or substantially the same width as the lateral flow region. In somecases, the reagent reservoir region is the same or substantially thesame height (i.e., thickness) as the lateral flow region. In otherembodiments, the reagent reservoir region is thicker than the lateralflow region, and therefore has a greater height (e.g., a greater heightin a non-compressed state). For example, the reagent reservoir regioncan be at least about 1.1, 1.2, 1.3, 1.5, 1.7, 2.0, 2.2, 2.5, 3-fold ormore thicker than the lateral flow region. In some embodiments, thereagent reservoir region is thinner than the lateral flow region.

In some embodiments, the reagent reservoir region has two or more (e.g.,at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or more) reagent reservoir sub-regions. In some cases, the two ormore reagent reservoir sub-regions enable detection of multipleanalytes. For example, one reagent reservoir sub-region can be used toreversibly immobilize a first primary binding reagent that detects afirst analyte and a second reagent reservoir sub-region can be used toreversibly immobilize a second primary binding reagent that detects asecond analyte, etc. In some cases, the number of reagent reservoirsub-regions determines the number of different analytes that can bedetected in a single blotting experiment. For example, a differentprimary binding reagent that specifically binds to a different analytecan be contacted with, or present in, each reagent reservoir sub-region.Alternatively, if the analytes to be detected are expected to beirreversibly immobilized in different locations on a membrane, multipleprimary binding reagents (e.g., 2, 3, 4, or more) can be used in asingle reagent reservoir region or sub-region. Reagent reservoirsub-regions can be separated by hydrophobic or impermeable barriers sothat a detection reagent in one sub-region does not migrate, wick, orotherwise flow from one reagent reservoir sub-region into anotherreagent reservoir sub-region.

In some embodiments, porous substrates containing at least two or morereagent reservoir sub-regions have a corresponding number of lateralflow sub-regions. In some cases, the corresponding lateral flowsub-regions are not in fluid communication until initiation of lateralflow. For example, the reagent reservoir and lateral flow sub regionscan be separated from their corresponding sub-regions by a hydrophobicor impermeable barrier. Thus, upon initiation of lateral flow, thebinding reagent, or a portion thereof, in each reagent reservoirsub-region flows through at least a portion of the corresponding lateralflow sub-region. Lateral flow sub-regions can be separated byhydrophobic or impermeable barriers so that a primary or secondarybinding reagent in one sub-region does not migrate, wick, or otherwiseflow from one lateral flow sub-region into another lateral flowsub-region.

FIG. 1 is an exemplary embodiment of a porous substrate having a reagentreservoir region and a lateral flow region, each region separated into anumber of sub-regions by hydrophobic barriers. In FIG. 1, the reagentreservoir region is loaded with three different binding reagents, eachseparated from each other by a hydrophobic barrier. The reagentreservoir regions are also each separated from their correspondinglateral flow sub-region by a hydrophobic barrier. As shown in FIG. 2,contacting the reagent reservoir region to the lateral flow region(e.g., by folding) initiates lateral flow. Thus, as depicted, at leastthree different membrane-bound analytes can be detected (e.g., inparallel) by the three different binding reagents. However, it will beappreciated that a number other than three (e.g., 2, 4, 5, 6, 7, 8,etc.) can be similarly run in parallel.

In some cases, the porous substrate contains a liquid sink. A liquidsink is a component of the porous substrate having a liquid absorbingcapacity that is significantly larger than that of the lateral flowregion. In some embodiments, the liquid sink is configured such that,upon initiation of lateral flow, liquid flows from the reagent reservoirregion to the lateral flow region and into the liquid sink. In somecases, the liquid sink is an integral component of the porous substrate.In some cases, the liquid sink is a distinct component of the poroussubstrate. In some cases, the liquid sink is configured or comprised ofmaterial that facilitates the rate of lateral flow through the lateralflow region. In some embodiments, the liquid sink is comprised of thesame material as the lateral flow region. In some embodiments, theliquid sink is comprised of different materials as compared to thelateral flow region. The liquid sink can be comprised of any of theporous substrate materials described herein, or a combination thereof.In some cases, the liquid sink is thicker (e.g., at least about 1.1,1.2, 1.3, 1.5, 1.7, 2.0, 2.2, 2.5, 3-fold or more thicker than thelateral flow region).

ii. Folding Region

In some embodiments, the porous substrate contains a folding region. Ingeneral, the folding region is situated and configured to allowcontacting the reagent reservoir region, or a portion thereof, with thelateral flow region, or a portion thereof, thereby initiating lateralflow.

The folding region can extend the width of the porous substrate or aportion thereof. In some cases, the folding region lies within, oradjacent to the reagent reservoir region. In some cases, the foldingregion lies within, or adjacent to the lateral flow region. In somecases, the folding region lies between the reagent reservoir region andthe lateral flow region. In some cases, the folding region lies withinor adjacent to an impermeable or hydrophobic barrier between the reagentreservoir region and the lateral flow region. In some cases, the foldingregion is demarcated. In some cases, the demarcation is a visual line orother mark identifying the location or approximate location of thefolding region. In some cases, the demarcation is a crease or pleat ofthe porous substrate.

iii. Barriers

The porous substrate can contain one or more barriers, such as one ormore hydrophobic barriers or one or more impermeable barriers. In somecases, the porous substrate has one or more barriers between a reagentreservoir region and a lateral flow region. In some cases, the poroussubstrate has one or more barriers between a reagent reservoirsub-region and another reagent reservoir sub-region. In some cases, theporous substrate has one or more barriers between a lateral flowsub-region and another lateral flow sub-region. In some cases, theporous substrate has one or more barriers between one or more reagentreservoir sub-regions and one or more lateral flow sub-regions. In someembodiments, one or more porous substrate barriers inhibit, eliminate,or substantially eliminate fluid communication (e.g., flow) betweenadjacent regions or sub-regions. In some cases, one or more poroussubstrate barriers inhibit, eliminate, or substantially eliminate fluidcommunication (e.g., flow) between adjacent regions or sub-regions untillateral flow is initiated.

Suitable barriers include hydrophobic barriers such as wax barriers, orbarriers created by vapor or liquid phase silanization of the poroussubstrate. Suitable barriers also include impermeable barriers, such asbarriers comprising a plastic, polymer, or resin. In some cases, theimpermeable barriers are frames that support a hydrophilic transfersheet suspended within the frame. In some cases, the barriers arecomprised of a combination of hydrophobic (e.g., wax or silanizedcellulose) and impermeable barriers. For example, in some cases, theporous substrates are surrounded by impermeable (e.g., plastic or resin)barrier frames and are further subdivided by wax or silanized cellulosebarriers.

The wax used to form the wax barriers can be any wax that is flowable atelevated temperatures and non-flowable at ambient temperature (e.g.,about 20-25° C.). Examples are paraffin waxes, microcrystalline waxes,thermoset waxes, animal waxes such as beeswax, lanolin, and tallow,vegetable waxes such as soy, carnauba, candelilla and palm waxes,mineral waxes such as ceresin and montan waxes, petroleum waxes, andsynthetic waxes such as ethylenic polymers, chlorinated naphthalenes,and Fischer-Tropsch waxes. Paraffin wax compositions may contain, inaddition to n-paraffins and isoparaffins, minor amounts ofcyclo-paraffins or olefins, or both. Waxes that become flowable, i.e.,that have melting points, within the temperature range of from about 60°C. to about 150° C., or from about 75° C. to about 125° C., are amongthose that can be used. Wax formulations and compositions that behave inthis manner are known to those of skill in the art.

The silanization reagent used to form hydrophobic barriers can be anysilanization reagent that reacts with the porous substrate, or a portionthereof. For example, if the porous substrate contains cellulose, asilanization reagent that silanizes hydroxyl groups of the cellulosebackbone can be utilized. Exemplary silanization reagents include, butare not limited to, trimethylchlorosilane, trimethylsilane, orhexamethyldisilazane. Silanization reagents further includetriethoxysilanes (R—Si(C₂H₅O)₃) where R is, for example, vinyl,methacrylol, aminopropyl, fluoroalkyl, or thioethyl. Other suitablesilanization reagents will be readily apparent to those of skill in theart.

The wax or other barrier forming reagent (e.g., silanization reagent, orimpermeable barrier) can be applied to one side or both sides of theporous substrate, although in most cases, application to one side willbe sufficient provided that the porous substrate penetrates, or is madeto penetrate (e.g., by melting after application), the porous substrateto a degree sufficient to serve as a barrier to the flow of liquid. Thebarrier forming reagent can be applied as a liquid. The liquid can beapplied by hand or other apparatus. In some cases, the liquid is sprayedor poured onto the porous substrate. Spraying can be accomplished withan inkjet printer or similar apparatus. In some cases, the liquidhardens after application to form an impermeable and/or hydrophobicbarrier. Alternatively, the barrier forming reagent can be applied as avapor. For example, a silanization reagent, wax, plastic, resin, orpolymer can be applied as a vapor that condenses on the porous substrateor reacts with the porous substrate. Alternatively, the barrier formingreagent can be applied as a solid. For example, wax can be applied as asolid manually or in an automated or mechanized fashion. In some cases,the porous substrate is masked to protect regions from the barrierforming reagent, and the barrier forming reagent is contacted with theporous substrate.

Application of wax can be achieved by hand, either by the use of acommon crayon or by a wax pen, or by a wax printer. Wax pens are knownin the art and commonly include a housing having a reservoir to containhot wax, a spout, and a handle. Application of the hot wax is achievedby tipping the housing to cause the liquefied wax to pass through thespout, and the housing is equipped with a valve to stop the flow of thewax at the terminus of a printed line. Wax printers are likewise knownin the art and commonly operated by thermal transfer printing using aprint head that includes an array of very small heating elements thatare software-controlled for independent activation to produce localizedheating of the wax above its melting point to release the wax to theprint medium. Commercially available examples of wax printers includethe Phaser 8560DN (Fuji Xerox, Ltd., Japan), and the CALCOMP COLORMASTERPLUS thermal wax transfer printer (CalComp Graphics, LLC, FoothillRanch, Calif., USA). Descriptions of wax printers and their use can befound in Kroon (Tektronix, Inc.), U.S. Pat. No. 5,957,593 (Sep. 28,1999); Lin (Xerox Corporation), U.S. Pat. No. 8,206,664 (Jun. 26, 2012);Lu, Y., et al., “Rapid prototyping of paper-based microfluidics with waxfor low-cost, portable bioassay,” Electrophoresis 2009, 30, 1497-1500;and Carrilho, E., et al., “Understanding Wax Printing: A SimpleMicropatterning Process for Paper-Based Microfluidics,” Anal. Chem.,2009, 81 (16), 7091-7095. The width of a wax line as applied (prior toheating) can vary and is not critical to the present invention, providedthat the amount of wax contained within the line is sufficient topenetrate the porous substrate and form a barrier to the lateral flow offluid within the porous substrate.

In some embodiments, once applied, the wax can be made to penetrate thebulk thickness of the porous substrate to fill the pores and form alateral barrier to aqueous fluid flow by heating the wax above itsmelting point. In some cases, the amount of wax applied will be suchthat full penetration of the thickness of the sheet with the melted waxwill occur while lateral flow of the melted wax (i.e., in directionsparallel to the flat faces of the sheet) is minimal or at least limitedto a small distance that is substantially uniform along the length of aline of applied wax so that the resulting area bordered by the waxbarrier is known and controlled. The formation of the barrier in thismanner can also be controlled by the degree of heating, including thetemperature to which the wax is heated and the length of time that theheating is continued. Optimal temperatures and durations are readilydeterminable by routine trial and error, but in most cases serviceableresults will be obtained by heating to at least 5° C. above the waxmelting point, and in many cases from about 5 to about 50° C. above themelting point, or from about 10 to about 30° C. above the melting point.The most appropriate heating time will depend on the temperature, highertemperatures requiring less time. In general, heating times ranging fromabout fifteen seconds to about twenty minutes, or in many cases fromabout thirty seconds to about ten minutes, will provide useful results.Heating can be achieved by conventional means, including radiativeheating, conductive heating, convective heating, impulse heating, andmicrowave heating. Effective results can be achieved with equipment assimple as a hot plate or a conventional oven.

Optimal widths for hydrophobic or impermeable barriers may vary with thedimensions of the area to be bordered by the barrier and with thethickness of the porous substrate and are readily determinable byroutine testing. In most cases, the width will range from about 10microns to about 5 mm, from about thirty microns to about 3 mm, fromabout 100 microns to about 1 mm, or from about 200 microns to about 5mm, or 10 mm.

B. Detection Reagents

i. Binding Reagents

Binding reagents are described herein for detection of analytes. In somecases, the binding reagents are antibodies (e.g., primary or secondaryantibodies). Primary antibodies can be used to bind to an analyte. Insome cases, the primary antibody is labeled enabling detection of theprimary antibody and consequently detection of the analyte. In somecases, the primary antibody is detected by binding to a labeledsecondary binding reagent, such as a labeled secondary antibody. In somecases, tertiary binding reagents are utilized to detect complexescontaining the analyte and the primary and secondary binding reagent.

In some embodiments, the porous substrate contains multiple (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) reagent reservoirsub-regions, and a corresponding number of binding reagents (e.g.,primary binding reagents such as primary antibodies). In some cases, theporous substrate contains multiple primary binding reagents and theprimary binding reagents are different in that they each detect adifferent analyte. In some cases, one or more of the primary bindingreagents are the same in that they detect the same analyte. In somecases, the porous substrate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, or 14 fewer binding reagents than reagent reservoir sub-regions.In some cases, a porous substrate containing multiple reagent reservoirsub-regions is provided to a customer or end-user and one or morebinding reagents are contacted to the reagent reservoir by the customeror end-user.

Binding reagents can be provided on or in the porous substrate or can besupplied separately. In some cases, a porous substrate contains one ormore binding reagents dried thereon. The dried binding reagent(s) can bereconstituted by contacting the reagent reservoir region with an aqueoussolution. In some cases, the aqueous reconstitution buffer can containone or more re-wetting reagents including salts, buffers, or a proteinaggregation modifying agent as described herein. Alternatively, thebinding reagent can be present in the porous substrate in a solution. Insome cases, the binding reagent(s) are stored in the porous substrate(e.g., the reagent reservoir region). For example, binding reagent(s)can be stored dry, substantially dry, or in solution in the poroussubstrate for at least about a day, three days, 7-10 days, at leastabout a month, two months, 3 months, six months, a year or longer. Insome cases, the binding reagents and porous substrate are suitable forstorage (e.g., at about 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 25,30, 35, or 37° C. or more) for at least about a day, three days, 7-10days, at least about a month, two months, 3 months, six months, a yearor longer.

ii. Labels

Analytes can be detected by detecting a label that is linked to abinding reagent. The label can be linked directly to the binding reagent(e.g., by a covalent bond) or the attachment can be indirect (e.g.,using a chelator or linker molecule). The terms “label” and “detectablelabel” are used synonymously herein. In some embodiments, each label(e.g., a first label linked to a first binding reagent, a second labellinked to a second binding reagent, etc.) generates a detectable signaland the signals (e.g., a first signal generated by the first label, asecond signal generated by the second label, etc.) are distinguishable.In some embodiments, the two or more binding reagent labels comprise thesame type of agent (e.g., a first label that is a first fluorescentagent and a second label that is a second fluorescent agent). In someembodiments, the two or more binding reagent labels (e.g., the firstlabel, second label, etc.) combine to produce a detectable signal thatis not generated in the absence of one or more of the labels.

Examples of detectable labels include, but are not limited to,biotin/streptavidin labels, nucleic acid (e.g., oligonucleotide) labels,chemically reactive labels, fluorescent labels, enzyme labels,radioactive labels, quantum dots, polymer dots, mass labels, colloidalgold, and combinations thereof. In some embodiments, the label caninclude an optical agent such as a chromophore, fluorescent agent,phosphorescent agent, chemiluminescent agent, etc. Numerous agents(e.g., dyes, probes, or indicators) are known in the art and can be usedin the present invention. (See, e.g., Invitrogen, The Handbook—A Guideto Fluorescent Probes and Labeling Technologies, Tenth Edition (2005)).Chromophores include co-enzymes or co-factors that have a detectableabsorbance. In some cases, a binding reagent can be detected bydetecting the intrinsic absorbance of a peptide bond at, e.g., 220 or280 nm.

Fluorescent agents can include a variety of organic and/or inorganicsmall molecules or a variety of fluorescent proteins and derivativesthereof. For example, fluorescent agents can include but are not limitedto cyanines, phthalocyanines, porphyrins, indocyanines, rhodamines,phenoxazines, phenylxanthenes, phenothiazines, phenoselenazines,fluoresceins (e.g., FITC, 5-carboxyfluorescein, and6-carboxyfluorescein), benzoporphyrins, squaraines, dipyrrolopyrimidones, tetracenes, quinolines, pyrazines, corrins, croconiums,acridones, phenanthridines, rhodamines (e.g., TAMRA, TMR, and RhodamineRed), acridines, anthraquinones, chalcogenopyrylium analogues, chlorins,naphthalocyanines, methine dyes, indolenium dyes, azo compounds,azulenes, azaazulenes, triphenyl methane dyes, indoles, benzoindoles,indocarbocyanines, benzoindocarbocyanines, BODIPY™ and BODIPY™derivatives, and analogs thereof. In some embodiments, a fluorescentagent is an Alexa Fluor dye. In some embodiments, a fluorescent agent isa polymer dot or a quantum dot. Fluorescent dyes and fluorescent labelreagents include those which are commercially available, e.g., fromInvitrogen/Molecular Probes (Eugene, Oreg.) and Pierce Biotechnology,Inc. (Rockford, Ill.). In some embodiments, the optical agent is anintercalating dye. In some embodiments, 2, 3, 4, 5, or more bindingreagents are each labeled with an optical agent such as a fluorescentagent (e.g., a first binding reagent labeled with a first fluorescentlabel, a second binding reagent labeled with a second fluorescent label,etc.), and each binding reagent that is labeled with an optical agent isdetected by detecting a signal generated by the optical agent (e.g., afluorescent signal generated by a fluorescent label). In someembodiments, all of the binding reagents are labeled with an opticalagent, and each optical agent-labeled binding reagent is detected bydetecting a signal generated by the optical agent.

In some embodiments, the label is a radioisotope. Radioisotopes includeradionuclides that emit gamma rays, positrons, beta and alpha particles,and X-rays. Suitable radionuclides include but are not limited to ²²⁵Ac,⁷²As, ²¹¹At, ¹¹B, ¹²⁸Ba, ²¹²Bi, ⁷⁵Br, ¹⁴C, ¹⁰⁹Cd, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ¹⁸F,⁶⁷Ga, ⁶⁸Ga, ³H, ¹⁶⁶Ho, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³⁰I, ¹³¹I, ¹¹¹In, ¹⁷⁷Lu, ¹³N,¹⁵O, ³²P, ³³P, ²¹²Pb, ¹⁰³Pd, ¹⁸⁶Re, ¹⁸⁸Re, ⁴⁷Sc, ¹⁵³Sm, ⁸⁹Sr, ^(99m)Tc,⁸⁸Y and ⁹⁰Y. In some embodiments, 2, 3, 4, 5 or, more binding reagentsare each labeled with a radioisotope (e.g., a first binding reagentlabeled with a first radioisotope, a second binding reagent labeled witha second radioisotope, etc.), and each binding reagent that is labeledwith a radioisotope is detected by detecting radioactivity generated bythe radioisotope. For example, one binding reagent can be labeled with agamma emitter and one binding reagent can be labeled with a betaemitter. Alternatively, the binding reagents can be labeled withradionuclides that emit the same particle (e.g., alpha, beta, or gamma)at different energies, where the different energies are distinguishable.In some embodiments, all of the binding reagents are labeled with aradioisotope and each labeled binding reagent can be detected bydetecting radioactivity generated by the radioisotope.

In some embodiments, the label is an enzyme, and the binding reagent isdetected by detecting a product generated by the enzyme. Examples ofsuitable enzymes include, but are not limited to, urease, alkalinephosphatase, (horseradish) hydrogen peroxidase (HRP), glucose oxidase,β-galactosidase, luciferase, alkaline phosphatase, and an esterase thathydrolyzes fluorescein diacetate. For example, a horseradish-peroxidasedetection system can be used with the chromogenic substratetetramethylbenzidine (TMB), which yields a soluble product in thepresence of hydrogen peroxide that is detectable at 450 nm. An alkalinephosphatase detection system can be used with the chromogenic substratep-nitrophenyl phosphate, which yields a soluble product readilydetectable at 405 nm. A β-galactosidase detection system can be usedwith the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside(ONPG), which yields a soluble product detectable at 410 nm. A ureasedetection system can be used with a substrate such as urea-bromocresolpurple (Sigma Immunochemicals; St. Louis, Mo.). In some embodiments, 2,3, 4, 5, or more binding reagents are each labeled with an enzyme (e.g.,a first binding reagent labeled with a first enzyme, a second bindingreagent labeled with a second enzyme, etc.), and each binding reagentthat is labeled with an enzyme is detected by detecting a productgenerated by the enzyme. In some embodiments, all of the bindingreagents are labeled with an enzyme, and each enzyme-labeled bindingreagent is detected by detecting a product generated by the enzyme.

In some embodiments, the label is an affinity tag. Examples of suitableaffinity tags include, but are not limited to, biotin, peptide tags(e.g., FLAG-tag, HA-tag, His-tag, Myc-tag, 5-tag, SBP-tag, Strep-tag,eXact-tag), and protein tags (e.g., GST-tag, MBP-tag, GFP-tag).

In some embodiments, the label is a nucleic acid label. Examples ofsuitable nucleic acid labels include, but are not limited to,oligonucleotide sequences, single-stranded DNA, double-stranded DNA, RNA(e.g., mRNA or miRNA), or DNA-RNA hybrids. In some embodiments, thenucleic acid label is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,or 1000 nucleotides in length.

In some embodiments, the label is a nucleic acid barcode. As used hereina “barcode” is a short nucleotide sequence (e.g., at least about 4, 6,8, 10, or 12, nucleotides long) that uniquely defines a labeledmolecule, or a second molecule bound to the labeled binding reagent. Thelength of the barcode sequence determines how many unique samples can bedifferentiated. For example, a 4 nucleotide barcode can differentiate 4⁴or 256 samples or less, a 6 nucleotide barcode can differentiate 4096different samples or less, and an 8 nucleotide barcode can index 65,536different samples or less. The use of barcode technology is well knownin the art, see for example Katsuyuki Shiroguchi, et al. Digital RNAsequencing minimizes sequence-dependent bias and amplification noisewith optimized single-molecule barcodes, PNAS (2012); and Smith, A M etal. Highly-multiplexed barcode sequencing: an efficient method forparallel analysis of pooled samples, Nucleic Acids Research Can 11,(2010).

In some embodiments, the label is a “click” chemistry moiety. Clickchemistry uses simple, robust reactions, such as the copper-catalyzedcycloaddition of azides and alkynes, to create intermolecular linkages.For a review of click chemistry, see Kolb et al., Agnew Chem40:2004-2021 (2001). In some embodiments, a click chemistry moiety(e.g., an azide or alkyne moiety) can be detected using anotherdetectable label (e.g., a fluorescently labeled, biotinylated, orradiolabeled alkyne or azide moiety).

Techniques for attaching detectable labels to binding reagents such asproteins (e.g., antibodies) are well known. For example, a review ofcommon protein labeling techniques can be found in BiochemicalTechniques: Theory and Practice, John F. Robyt and Bernard J. White,Waveland Press, Inc. (1987). Other labeling techniques are reviewed in,e.g., R. Haugland, Excited States of Biopolymers, Steiner ed., PlenumPress (1983); Fluorogenic Probe Design and Synthesis: A Technical Guide,PE Applied Biosystems (1996); and G. T. Herman, Bioconjugate Techniques,Academic Press (1996).

In some embodiments, two or more labels (e.g., a first label, secondlabel, etc.) combine to produce a detectable signal that is notgenerated in the absence of one or more of the labels. For example, insome embodiments, each of the labels is an enzyme, and the activities ofthe enzymes combine to generate a detectable signal that is indicativeof the presence of the labels (and thus, is indicative of each of thelabeled proteins). Examples of enzymes combining to generate adetectable signal include coupled assays, such as a coupled assay usinghexokinase and glucose-6-phosphate dehydrogenase; and a chemiluminescentassay for NAD(P)H coupled to a glucose-6-phosphate dehydrogenase,beta-D-galactosidase, or alkaline phosphatase assay. See, e.g., Maeda etal., J Biolumin Chemilumin 1989, 4:140-148.

C. Protein Aggregation Modifying Agents

Described herein are protein aggregation modifying agents. Proteinaggregation modifying agents can be utilized to reduce or eliminateaggregation or denaturation of binding reagents, such as proteins (e.g.,antibodies), stored on, or delivered from, a porous substrate. Forexample, protein aggregation modifying agents can be utilized to reduceor eliminate aggregation or denaturation of primary antibodies storedin, or delivered from, the reagent reservoir region of a poroussubstrate. In some cases, protein aggregation modifying agents can beutilized to facilitate lateral flow of binding reagents in the lateralflow region of the porous substrate.

In some cases, protein aggregation modifying agents that act to displaceproteins from the air-water interface and thereby protect them fromdenaturation and aggregation are particularly effective in reducing theaggregation of binding reagents immobilized on a porous substrate. Inother cases, the protein aggregation modifying agent directly affectsthe stability of the binding reagent by binding to the binding reagentand/or stabilizing the binding reagent. In other cases, the proteinaggregation modifying agent acts to shift the equilibrium away from adenatured or unfolded state and thus reduce aggregation. For example, insome cases, the interaction between the protein aggregation modifyingagent and the binding reagent is thermodynamically disfavored due tostrong repulsion between an amide backbone of the binding reagent andthe protein aggregation modifying agent. Thus, unfolding of the bindingreagent in the presence of the protein aggregation modifying agent isdisfavored because unfolding exposes more amide backbone surface to theprotein aggregation modifying agent.

Protein aggregation modifying agents can be one or more of acyclodextrin, a non-ionic surfactant, an ionic surfactant, azwitterionic surfactant, a non-detergent sulfobetaine, a simple sugar, apolysaccharide, a polyol, an organic solvent, an aggregation modifyingprotein, a disordered peptide sequence, an amino acid, anoxido-reduction agent, a lyoprotectant, a cryoprotectant, or achaotropic agent.

Cyclodextrins can be, but are not limited to, α-cyclodextrin,β-cyclodextrin, γ-cyclodextrin, (2,3,6-tri-O-methyl)-β-cyclodextrin,(2,3,6-tri-O-methyl)-β-cyclodextrin, (2-hydroxy)propyl-β-cyclodextrin,(2-hydroxy)propyl-γ-cyclodextrin, random methyl-β-cyclodextrin, randommethyl-γ-cyclodextrin, carboxymethyl-β-cyclodextrin,carboxymethyl-γ-cyclodextrin, 6-monodeoxy-6-monoamino-β-cyclodextrin,sulfobutyl-β-cyclodextrin, 6-amino-6-deoxy-β-cyclodextrin, acetylβ-cyclodextrin, succinyl α-cyclodextrin, succinyl β-cyclodextrin,succinyl γ-cyclodextrin, (2,3,6-tri-O-benzoyl)-β-cyclodextrin,succinyl-(2-hydroxypropyl)-β-cyclodextrin, orsuccinyl-(2-hydroxypropyl)-γ-cyclodextrin. Cyclodextrins can also be acyclodextrin polymer containing one or more of the foregoingcyclodextrin molecules. Additional cyclodextrins are known in the art,and include, e.g. those described on the world wide web atcyclodextrin.com. Exemplary concentrations of cyclodextrins are, withoutlimitation, about 1 mM, 2 mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM, 15 mM, 20 mM,25 mM, 50 mM, 75 mM, or 100 mM.

Non-ionic surfactants can be polyethylene-sorbitan-fatty acid esters,polyethylene-polypropylene glycols or polyoxyethylene-stearates.Polyethylene-sorbitan-fatty acid esters can bepolyethylene(20)-sorbitan-esters (Tween20™) orpolyoxyethylene(20)-sorbitanmonooleate (Tween 80™).Polyethylene-polypropylene glycols can bepolyoxypropylene-polyoxyethylene block co-polymers such as those soldunder the names Pluronic® or Poloxamer™ Polyoxyethylene-stearates canbe, for example, those sold under the trademark Myrj™ Exemplary,polyoxyethylene monolauryl ethers include those sold under the trademarkBrij™ e.g., Brij-35. Exemplary concentrations of non-ionic surfactantsare, without limitation, about 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%,0.75%, 1%, 2%, 2.5%, 5%, 7.5%, or about 10% w/w, w/v, or v/v.

Ionic surfactants can be anionic surfactants or cationic surfactants.Anionic surfactants useful in the present invention can be, but are notlimited to, soaps including alkali soaps, such as sodium, potassium orammonium salts of aliphatic carboxylic acids, usually fatty acids, suchas sodium stearate. Additional anionic surfactants include organic aminesoaps such as organic amine salts of aliphatic carboxylic acids, usuallyfatty acids, such as triethanolamine stearate. Cationic surfactantsuseful in the present invention include, but are not limited to, aminesalts such as octadecyl ammonium chloride or quarternary ammoniumcompounds such as benzalkonium chloride. Ionic surfactants can includethe sodium, potassium or ammonium salts of alkyl sulfates, such assodium dodecyl sulfate or sodium octyl sulfate. Exemplary concentrationsof ionic surfactants are, without limitation, about 0.01%, 0.02%, 0.05%,0.1%, 0.2%, 0.5%, 0.75%, 1%, 2%, 2.5%, 5%, 7.5%, or about 10% w/w, w/v,or v/v.

Zwitterionic surfactants have both cationic and anionic centers attachedto the same molecule. The cationic part is, e.g., based on primary,secondary, or tertiary amines or quaternary ammonium cations. Theanionic part can be a sulfonate, as in CHAPS(3[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate). Otheranionic groups are sultaines illustrated by cocamidopropylhydroxysultaine or betaines, e.g., cocamidoethyl betaine, cocamidopropylbetaine, or lauramidopropyl betaine. Exemplary concentrations ofzwitterionic surfactants are, without limitation, about 0.01%, 0.02%,0.05%, 0.1%, 0.2%, 0.5%, 0.75%, 1%, 2%, 2.5%, 5%, 7.5%, and about 10%w/w, w/v, or v/v.

Non detergent sulfobetaines (NDSBs) have a sulfobetaine hydrophilicgroup and a short hydrophobic group that cannot aggregate to formmicelles, therefore NDSBs are not considered detergents. Exemplary NDSBsinclude, but are not limited to NDSB 256, NDSB 221, NDSB 211, NDSB 201,NDSB 195, 3-(4-tert-Butyl-1-pyridinio)-1-propanesulfonate,3-(1-pyridinio)-1-propanesulfonate, 3-(Benzyldimethylammonio)propanesulfonate, or Dimethylethylammoniumpropane sulfonate. Exemplaryconcentrations of NDSBs include, but are not limited to about 0.01%,0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.75%, 1%, 2%, 2.5%, 5%, 7.5%, and about10% w/w, w/v, or v/v.

Polyols are compounds with multiple hydroxyl functional groups. In somecases, polyols can modify the aggregation or denaturation behavior of aprotein by a variety of mechanisms. For example, in some cases, thepolyol can shift the equilibrium to the folded state by presenting athermodynamically disfavored interaction with the protein backbone.Alternatively, in some cases, the polyol can bind to and stabilize thefolded state of the protein.

Polyols can be simple sugars such as sucrose, mannitol, sorbitol,inositol, xylitol, erythritol, glucose, galactose, raffinose, ortrehalose. Polyols can also be polysaccharides such as dextran, starch,hydroxyethyl starch, or polymers containing one or more of the simplesugars described herein. Glycerol, ethylene glycol, polyethylene glycol,pentaerythritol propoxylate, and pentaerythritol propoxylate, andcombinations thereof are also exemplary polyols.

Organic solvents can be, but are not limited to, those organic solventthat are known to inhibit denaturation, unfolding, or aggregation of oneor more proteins. A variety of suitable organic solvents are known inthe art. For example, organic solvents can include ethanol, butanol,propanol, phenol, dimethyl formamide, 2-methyl-2,4-pentanediol,2,3-butanediol, 1,2-propanediol, 1,6-hexanediol, or dimethyl sulfoxide.

Aggregation modifying proteins can be proteins known in the art toinhibit denaturation, unfolding, or aggregation of one or more proteins.Exemplary aggregation modifying proteins include, but are not limitedto, albumins. Albumins are proteins that are water-soluble, aremoderately soluble in concentrated salt solutions, and experience heatdenaturation. Exemplary albumins include serum albumins (e.g., bovine,horse, or human serum albumin) or egg albumin (e.g., hen egg-whitealbumin). Other exemplary aggregation modifying proteins include casein,gelatin, ubiquitin, lysozyme, or late embryogenesis abundant (LEA)proteins. LEA proteins include LEA I, LEA II, LEA III, LEA IV, LEA V, oratypical LEA proteins. LEA proteins are known in the art and described,e.g., in Goyal K., et al., Biochemical Journal 288(pt. 1), 151-57,(2005).

Protein aggregation modifying agents can also be amino acids. In somecases, the amino acids can serve an oxido-reduction function to maintainan appropriate oxidative potential for the protein immobilized on thesubstrate. Suitable oxido-reductive amino acids include cysteine andcystine. Other amino acids serve to reduce denaturation or aggregationthrough a non-oxido-reductive method. For example, arginine, glycine,proline, and taurine have been shown to reduce protein aggregation.

Other oxido-reduction agents can be employed to reduce proteinaggregation. Oxido-reductants other than cysteine and cystine, can beused to optimize the reduction potential in the substrate onto which theprotein is immobilized. Exemplary oxido-reductants includemercaptoethanol, dithiothreitol, dithioerythritol,tris(2-carboxyethyl)phosphine, glutathione, glutathione disulfide, andoxidized derivatives thereof, as well as Cu²⁺.

Protein aggregation modifying agents can also include lyoprotectants,cryoprotectants, or chaotropic agents. In some cases, the proteinaggregation modifying agent is a chaotrope such as urea, thiourea,guanidinium, cyanate, thiocyanate, trimethylammonium,tetramethylammonium, cesium, rubidium, nitrate, acetate, iodide,bromide, trichloroacetate, or perchlorate. Under certain conditions,such as at low concentrations, chaotropes can reduce proteinaggregation. Other protein aggregation modifying agents includetrimethylamine N-oxide.

Protein aggregation modifying agents can be salts. Exemplary saltsinclude, but not limited to, the sodium, potassium, magnesium, orcalcium salts of chloride, sulfate, or phosphate. Protein aggregationmodifying agents can also be buffering agents. Exemplary bufferingagents include, but are not limited to, tris (hydroxymethyl) aminomethene (TRIS), TAPSO, IVIES, HEPES, PIPES, CAPS, CAPSO, MOPS, MOPSO, orsodium or potassium phosphate, carbonate, bicarbonate, citrate, acetate,or borate buffers.

The protein aggregation modifying agents can be provided in any suitableconcentration. In some cases, the protein is provided as an aqueoussolution containing binding reagent and protein aggregation modifyingagents. In such cases, the solution can be contacted with a poroussubstrate and, optionally, dried. Exemplary concentrations of proteinaggregation modifying agents in the aqueous binding reagent solutioninclude, but are not limited to, about 0.001%, 0.005%, 0.01%, 0.05%,0.1%, 0.5%, 1%, 2%, 4%, 5%, 10%, 20%, or about 25% or more w/v of thesolution. Further exemplary concentrations include, but are not limitedto, about 1 μM, 5 μM, 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, 150 μM, 200μM, 300 μM, 500 μM, 750 μM, 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 150mM, 200 mM, 300 mM, 500 mM, and 1M.

In some cases, the protein aggregation modifying agents are provided onthe porous substrate. Exemplary compositions containing a proteinaggregation modifying agent and a porous substrate include, substrates,or regions therein (e.g., a reagent reservoir region) that contain about0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or about10%, 20%, or about 25% by weight of one or more protein aggregationmodifying agents.

Protein aggregation modifying agents can be provided in any suitablecombination. For example, in some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more of the foregoing protein aggregation modifying agents can beutilized to reduce aggregation of a binding reagent reversiblyimmobilized on a porous substrate. In some cases, prior to contactingthe porous substrate with the binding reagent solution, the poroussubstrate contains a protein aggregation modifying agent, and thebinding reagent solution contains the same, or a different, proteinaggregation modifying agent. In some cases, prior to contacting thesubstrate with the binding reagent solution, the substrate contains aprotein aggregation modifying agent, and the binding reagent solutiondoes not contain a protein aggregation modifying agent. In some cases,prior to contacting the substrate with the binding reagent solution, thebinding reagent solution contains a protein aggregation modifying agentand the substrate, or the region to be contacted, does not.

IV. Methods

Compositions and methods described herein can be utilized to detect oneor more analytes. For example, compositions and methods can be utilizedto detect one or more analytes that are irreversibly bound to amembrane. In some cases, compositions and methods described herein canbe utilized to perform a blotting experiment.

An exemplary embodiment is depicted in FIG. 3. FIG. 3 depicts multiplexdetection of three different analytes using compositions and methodsdescribed herein. In FIG. 3, step 1, three different binding reagents(e.g., antibodies) are loaded onto three different reagent reservoirsub-regions of a porous substrate. The reagent reservoir sub-regions areseparated from each other and from the lateral flow region byhydrophobic or impermeable barriers. The porous substrate can be placedin intimate contact with a membrane containing bound (e.g., irreversiblybound) analytes before or after the loading step. In step 2, the reagentreservoir region is contacted with the lateral flow region to initiatelateral flow by, e.g., folding. Lateral flow of the binding reagents intheir corresponding lateral flow sub-regions thus allows the bindingreagents to bind, and thereby detect, membrane-bound analytes. Thus, inthis embodiment, at least three different membrane-bound analytes can bedetected simultaneously.

A membrane containing one or more irreversibly bound analyte(s) can beplaced in intimate contact with a porous substrate, such as a poroussubstrate having a lateral flow region. Subsequently initiating flow ofone or more binding reagents through the porous substrate can thencontact the binding reagent(s) to the membrane-bound analyte(s). In someembodiments, the initiating of lateral flow of binding reagent(s)through the porous substrate is performed by contacting the poroussubstrate, or contacting the lateral flow region of the poroussubstrate, or a portion thereof, with a reagent reservoir region, orportion thereof.

The membrane and/or the lateral flow region can be blocked with ablocking agent before initiating lateral flow of the binding reagent. Insome cases, the blocking is performed before placing the membrane inintimate contact with the porous substrate. Alternatively, the blockingis performed after placing the membrane in intimate contact with theporous substrate. Blocking agents are well known in the art and can beused to reduce or eliminate non-specific binding of one or more bindingreagents (e.g., one or more primary or secondary binding reagents).Exemplary blocking agents include, without limitation, bovine serumalbumin (BSA), methylated BSA, casein, nonfat dry milk, serum, gelatin,or a protein free blocking solution. Exemplary protein free blockingsolutions include solutions containing hydrophilic or amphiphilicsynthetic polymers.

The lateral flow region and the reagent reservoir region of the poroussubstrate can be separated by a folding region. In some cases, thecontacting of the reagent reservoir region to the lateral flow region isperformed by folding the porous substrate at the folding region. Foldingthe porous substrate at the folding region can contact at least aportion of the reagent reservoir region to at least a portion of thelateral flow region and thus initiate lateral flow of one or morebinding reagents from the reagent reservoir region and through thelateral flow region.

The reagent reservoir region can be integral to the porous substrate.Alternatively, the reagent reservoir region can be distinct from, butattached to, the porous substrate (e.g., attached to the lateral flowregion of the porous substrate). In still other cases, the reagentreservoir region can be a second porous substrate that is contacted witha first porous substrate in intimate contact with the membrane. In someembodiments, the reagent reservoir region contains multiple reagentreservoir sub-regions. In some cases, the reagent reservoir sub-regionscan contain different binding reagents.

The reagent reservoir can contain an aqueous solution containing bindingreagent(s). For example, the porous substrate can be provided withbinding reagents dried thereon, the binding reagents reconstituted withan aqueous buffered solution, and then used as described herein todetect one or more analytes. The reconstitution can be performed beforeor after placing the porous substrate in intimate contact with themembrane. Alternatively, the porous substrate can be provided with anaqueous solution containing binding reagents and used as describedherein. As yet another alternative, the binding reagents can be appliedby the end-user as a solution to the reagent reservoir region,optionally dried and then reconstituted, and then used as describedherein.

In some embodiments, upon contacting the reagent reservoir region to thelateral flow region, a binding reagent, or portion thereof, (e.g., asolution containing a binding reagent, or a portion thereof) is wickedaway from the reagent reservoir region and through the lateral flowregion. In some cases, the contacting causes the binding reagent, orportion thereof to be wicked away from the reagent reservoir region,through the lateral flow region, and into a liquid sink.

The method can include one or more blocking, washing, or secondarydetection steps. In some cases, one or more blocking, washing, orsecondary detection steps is performed by sequential lateral flow.Sequential lateral flow refers to performing multiple lateral flow stepsin a single porous substrate. For example, a porous substrate can beplaced in intimate contact with a membrane containing one or moreirreversibly bound analytes. Optionally, lateral flow of a wash and/orblocking solution through the porous substrate can then be initiated.Alternatively, or additionally, the membrane can be washed and/orblocked prior to being placed in intimate contact with the poroussubstrate. Lateral flow of one or more primary binding reagents throughthe porous substrate (e.g., through a lateral flow region therein) canbe initiated, thereby contacting the binding reagent to the irreversiblybound analyte(s). Lateral flow of a wash and/or blocking solutionthrough the porous substrate can then be initiated. In some cases, thewash solution can remove excess or unbound binding reagent. Lateral flowof one or more secondary binding reagents can then be initiated. Excessor unbound secondary binding reagents can be removed by a subsequentblocking or washing step (e.g., by sequential lateral flow).

Sequential lateral flow can be performed with an active device thatemploys one or more pumps, valves or motors. Alternatively, sequentiallateral flow can be performed with a passive device that does notutilize a pump, a valve, a motor, or require an electrical power source.Exemplary passive sequential lateral flow devices are described in U.S.Patent Application Nos. 2010/0239459 and 2013/0164193.

In some cases, one or more blocking or washing steps is performed bypouring, pipetting, or spraying a blocking or washing solution onto theporous substrate. Alternatively, the porous substrate can be soaked in ablocking or washing solution. The pouring, pipetting, spraying orsoaking can be performed manually or using an automated device.

In some embodiments, after contacting one or more primary bindingreagents (e.g., one or more primary antibodies) to the membrane bylateral flow through the porous substrate, the porous substrate can beremoved and one or more, or all, subsequent blocking, washing, orsecondary detection steps performed on the membrane.

One or more irreversibly bound analytes can be detected on the membraneby detecting a bound primary or secondary binding reagent (e.g.,detecting a labeled primary or secondary antibody). Methods fordetecting primary or secondary binding reagents bound to membrane-boundanalytes are well known in the art.

Porous substrates can be stored for a period of time before use. Forexample the porous substrate can be stored for at least about a day,three days, 7-10 days, at least about a month, two months, 3 months, sixmonths, a year or longer, e.g., at about 4, 5, 6, 7, 8, 10, 12, 14, 16,18, 20, 22, 25, 30, 35, or 37° C. or more. In some cases, the poroussubstrate is stored with one or more binding reagents therein/on. Thebinding reagents can be stored in or on the porous substrate dry,substantially dry, or wet. In some cases, the porous substrate or aportion thereof (e.g., the reagent reservoir region) is reconstitutedbefore use.

V. Kits

Kits for performing any of the foregoing methods are described herein.Also described herein are kits containing one or more of the foregoingcompositions. In some embodiments, the kit contains one or more (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) porous substrates. In someembodiments, the kit contains one or more binding reagents (e.g., one ormore primary and/or secondary binding reagents). In some cases, kitcontains one or more porous substrates with one or more binding reagentsreversibly bound therein. In some cases, the binding reagent(s) aredried onto the porous substrate, or a portion thereof (e.g., dried ontoa reservoir region or sub-region). In some cases, the binding reagentsare dried onto the porous substrate or portion thereof in the presenceof one or more protein aggregation modifying agents. In some cases, thebinding reagent(s) are provided as a reagent to be applied to the poroussubstrate by the end-user.

In some embodiments, the kit contains a cyclodextrin or other proteinaggregation modifying agent. In some cases the kit contains one or moreof the foregoing porous substrates impregnated with a proteinaggregation modifying agent. In some embodiments, the kit contains aprotein aggregation modifying agent that can be applied to a poroussubstrate, or a portion thereof, by the end-user. The proteinaggregation modifying agent of the kit can, for example, be provided asa solid (e.g., a powder) or in liquid form (e.g., as a solution). Insome cases, the kit contains a protein aggregation modifying agent thatcan be applied to a porous substrate (e.g., as part of an aqueoussolution), or a portion thereof, by the end-user during reconstitutionof one or more reversibly bound binding reagents.

VI. Examples

Hydrophobic barriers (e.g., wax barriers) are printed on a poroussubstrate (e.g., Whatman chromatography paper). The hydrophobic barriersseparate a binding reagent reservoir region from a lateral flow region.Hydrophobic barriers also separate reagent reservoir sub-regions.Similarly, hydrophobic barriers separate lateral flow sub-regions.Binding reagents (e.g., antibodies) are loaded onto one, two or threereagent reservoir sub-regions. The hydrophobic barriers block flowbetween the reagent reservoir and the lateral flow regions, betweenreagent reservoir sub-regions and between lateral flow sub-regions. See,FIG. 1.

The porous substrate is placed in intimate contact with a western blotmembrane containing bound proteins. Folding the porous substrate at thefolding region to contact the reagent reservoir region to the lateralflow region initiates lateral flow of the binding reagents through thelateral flow sub-regions. Binding reagents in each lateral flowsub-region do not mix during lateral flow due to the hydrophobicbarriers. See, FIG. 2. Thus, each sub-region can independently detectone or more analytes, enabling multiplex detection of analytes bound toa membrane. See, FIG. 3.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1-15. (canceled)
 16. A method for performing a lateral flow assaycomprising: providing a porous substrate comprising a lateral flowregion; placing the porous substrate in intimate contact with a membranecomprising a plurality of immobilized analytes; and contacting thelateral flow region with a reagent reservoir region comprising a primarybinding reagent, thereby causing at least a portion of the primarybinding reagent to wick into the lateral flow region, wherein causing atleast a portion of the primary binding reagent to wick into the lateralflow region initiates lateral flow of the primary binding reagentthrough at least a portion of the lateral flow region, therebycontacting at least a portion of the primary binding reagent to themembrane comprising a plurality of immobilized analytes.
 17. The methodof claim 16, the method further comprising initiating lateral flow of asolution containing a secondary binding reagent through at least aportion of the lateral flow region.
 18. The method of claim 17, themethod further comprising initiating lateral flow of a wash solutionafter initiating lateral flow of the solution containing a secondarybinding reagent through at least a portion of the lateral flow region.19. The method of claim 16, wherein the porous substrate comprising alateral flow region further comprises the reagent reservoir region, andwherein the reagent reservoir region is separated from the lateral flowregion by (i) an impermeable or hydrophobic barrier, and (ii) a foldingregion. 20-23. (canceled)
 24. The method of claim 16, the method furthercomprising initiating lateral flow of a wash solution through at least aportion of the lateral flow region.
 25. The method of claim 19, whereinthe contacting comprises folding the porous substrate at the foldingregion.
 26. The method of claim 19, wherein the folding region extendsacross the width of the porous substrate.
 27. The method of claim 25,wherein the lateral flow region is configured to wick one or morebinding reagents from the reagent reservoir and along the length of thelateral flow region after folding the folding region.
 28. The method ofclaim 19, wherein the folding region comprises a visual marker, a pleat,or a crease.
 29. The method of claim 16, wherein at least a portion ofthe lateral flow region is substantially compressible.
 30. The method ofclaim 16, wherein at least a portion of the reagent reservoir region issubstantially compressible.
 31. The method of claim 16, wherein at leasta portion of the porous substrate is substantially compressible.
 32. Themethod claim 16, wherein: said reagent reservoir region comprises atleast a first reagent reservoir sub-region and a second reagentreservoir sub-region, wherein the first and second reagent reservoirsub-regions are not in fluid communication; and said lateral flow regioncomprises at least a first lateral flow sub-region and a second lateralflow sub-region, wherein said first and second lateral flow sub-regionsare not in fluid communication.
 33. The method of claim 32, wherein thefirst reagent reservoir sub-region is separated from the second reagentreservoir sub-region by a hydrophobic or impermeable barrier, andwherein the first lateral flow sub-region is separated from the secondlateral flow sub-region by a hydrophobic or impermeable barrier.
 34. Themethod of claim 32, wherein the first reagent reservoir sub-regioncomprises a first primary binding reagent and the second reagentreservoir sub-region comprises a second primary binding reagent.
 35. Themethod of claim 16, wherein the reagent reservoir region comprises aprotein aggregation modifying agent.
 36. The method of claim 16, whereinthe lateral flow region comprises a capillary flow matrix comprisingcellulose or glass fibers.
 37. The method of claim 16, wherein thereagent reservoir region comprises a capillary flow matrix comprisingcellulose or glass fibers.
 38. The method of claim 36, wherein at leasta portion of the capillary flow matrix is coupled to an impermeable orhydrophobic backing.
 39. The method of claim 36, wherein the capillaryflow matrix comprises glass fibers.